Vaccinia-related Kinase Research Articles

The TFAMoplex-Conversion of the Mitochondrial Transcription Factor A into a DNA Transfection Agent.

Non‐viral gene delivery agents, such as cationic lipids, polymers, and peptides, mainly rely on charge‐based and hydrophobic interactions for the condensation of DNA molecules into nanoparticles. The human protein mitochondrial transcription factor A (TFAM), on the other hand, has evolved to form nanoparticles with DNA through highly specific protein‐protein and protein‐DNA interactions. Here, the properties of TFAM are repurposed to create a DNA transfection agent by means of protein engineering. TFAM is covalently fused to Listeria monocytogenes phospholipase C (PLC), an enzyme that lyses lipid membranes under acidic conditions, to enable endosomal escape and human vaccinia‐related kinase 1 (VRK1), which is intended to protect the DNA from cytoplasmic defense mechanisms. The TFAM/DNA complexes (TFAMoplexes) are stabilized by cysteine point mutations introduced rationally in the TFAM homodimerization site, resulting in particles, which show maximal activity when formed in 80% serum and transfect HeLa cells in vitro after 30 min of incubation under challenging cell culture conditions. The herein developed TFAM‐based DNA scaffolds combine interesting characteristics in an easy‐to‐use system and can be readily expanded with further protein factors. This makes the TFAMoplex a promising tool in protein‐based gene delivery.

VRK2 activates TNFα/NF-κB signaling by phosphorylating IKKβ in pancreatic cancer.

NF-κB signaling is active in more than 50% of patients with pancreatic cancer and plays an important role in promoting the progression of pancreatic cancer. Revealing the activation mechanism of NF-κB signaling is important for the treatment of pancreatic cancer. In this study, the regulation of TNFα/NF-κB signaling by VRK2 (vaccinia-related kinase 2) was investigated. The levels of VRK2 protein were examined by immunohistochemistry (IHC). The functions of VRK2 in the progression of pancreatic cancer were examined using CCK8 assay, anchorage-independent assay, EdU assay and tumorigenesis assay. The regulation of VRK2 on the NF-κB signaling was investigated by immunoprecipitation and invitro kinase assay. It was discovered in this study that the expression of VRK2 was upregulated in pancreatic cancer and that the VRK2 expression level was significantly correlated with the pathological characteristics and the survival time of patients. VRK2 promoted the growth, sphere formation and subcutaneous tumorigenesis of pancreatic carcinoma cells as well as the organoid growth derived from the pancreatic cancer mouse model. Investigation of the molecular mechanism indicated that VRK2 interacts with IKKβ, phosphorylating its Ser177 and Ser181 residues and thus activating the TNFα/NF-κB signaling pathway. An IKKβ inhibitors abolished the promotive effect of VRK2 on the growth of organoids. The findings of this study indicate that VRK2 promotes the progression of pancreatic cancer by activating the TNFα/NF-κB signaling pathway, suggesting that VRK2 is a potential therapeutic target for pancreatic cancer.

Purification, characterization and functional site prediction of the vaccinia-related kinase 2A small transmembrane domain

Vaccinia-related kinases (VRK) are serine-threonine kinases that regulate several signaling pathways. The isoform-VRK2A of one such kinase VRK2 controls cell stress response by interacting with TAK1, a mitogen-activated protein 3 kinase (MAP3K), via its partly cytosolic C-terminal transmembrane domain (VTMD). To establish the driving force and identify the key residues of the VRK2A-TAK1 interaction, we expressed and purified the standalone 3.6 kDa VTMD in the bacterial system using a unique and atypical two-step approach, when the effort to obtain full-length VRK2A remained unsuccessful. Characterization of biophysical properties demonstrated that VTMD domain maintains its structural integrity. Furthermore, dissecting the VRK2A-TAK1 binding interface using in silico tools provided important cues toward engineering the VRK2A-TAK1 interface to modulate its functions with desired characteristics. Most importantly, this novel purification strategy demonstrates its universal applicability in protein biochemistry research by serving as a model system for obtaining difficult-to-purify small proteins or domains.•VRK2A is a highly disordered transmembrane (TM) kinase, whose TM domain interacts with TAK1 (transforming growth factor-β-activated kinase).•The standalone VRK2A-TM domain (VTMD) was purified using affinity chromatography followed by two-step centricon based approach.•Biophysical and in silico analyses confirmed structural integrity of the domain.

Purification and Biophysical Characterization of the C-Terminal Transmembrane Domain of Vaccinia-Related Kinase 2a

Purification and Biophysical Characterization of the C-Terminal Transmembrane Domain of Vaccinia-Related Kinase 2a

Dysfunctional Homozygous VRK1-D263G Variant Impairs the Assembly of Cajal Bodies and DNA Damage Response in Hereditary Spastic Paraplegia.

Background and ObjectivesTo conduct a genetic and molecular functional study of a family with members affected of hereditary spastic paraplegia (HSP) of unknown origin and carrying a novel pathogenic vaccinia-related kinase 1 (VRK1) variant.MethodsWhole-exome sequencing was performed in 2 patients, and their parents diagnosed with HSP. The novel VRK1 variant was detected by whole-exome sequencing, molecularly modeled and biochemically characterized in kinase assays. Functionally, we studied the role of this VRK1 variant in DNA damage response and its effect on the assembly of Cajal bodies (CBs).ResultsWe have identified a very rare homozygous variant VRK1-D263G with a neurologic phenotype associated with HSP and moderate intellectual disability. The molecular modeling of this VRK1 variant protein predicted an alteration in the folding of a loop that interferes with the access to the kinase catalytic site. The VRK1-D263G variant is kinase inactive and does not phosphorylate histones H2AX and H3, transcription factors activating transcription factor 2 and p53, coilin needed for assembly of CBs, and p53 binding protein 1, a DNA repair protein. Functionally, this VRK1 variant protein impairs CB formation and the DNA damage response.DiscussionThis report expands the neurologic spectrum of neuromotor syndromes associated with a new and rare VRK1 variant, representing a novel pathogenic participant in complicated HSP and demonstrates that CBs and the DNA damage response are impaired in these patients.

O-118 New insight into the genetic contribution of common variants to the development of extreme phenotypes of unexplained male infertility: a multicenter genome-wide association study

Abstract Study question What is the contribution of the common genetic variation to the development of unexplained male infertility due to severe spermatogenic failure (SPGF)? Summary answer Genetic polymorphisms of key immune and spermatogenesis loci are involved in the etiology of the most severe SPGF cases, defined by Sertoli cell-only (SCO) phenotype. What is known already Male infertility is a rising worldwide concern that affects millions of couples. Non-obstructive azoospermia (NOA) and severe oligospermia (SO) are two extreme manifestations characterized by SPGF. A genetic cause can be established in only around 20% of affected men, with the remaining cases being classified as otherwise unexplained. To date, the genome-wide association study (GWAS) strategy, although already successfully applied in several other complex traits and diseases, was less fruitful in studies that attempted to decipher the genetic component of unexplained SPGF, mainly due to both a lack of well-powered samples in different ancestries and limitations in study design. Study design, size, duration We designed a GWAS for unexplained male infertility due to SPGF including a total of 1,274 affected cases and 1,951 fertile controls from the Iberian Peninsula (Spain and Portugal) and Germany. Different biostatistics and bioinformatics approaches were used to evaluate the possible effect of single-nucleotide polymorphisms (SNPs) across the whole genome in the susceptibility to specific subtypes of unexplained SPGF. Participants/materials, setting, methods The case cohort comprised 502 SO and 772 NOA patients, who were subdivided according to histological phenotypes (SCO, maturation arrest, and hypospermatogenesis) and the outcome of testicular sperm extraction techniques (TESE) from testis biopsies. Genotyping was performed with the GSA platform (Illumina). After quality-control and genotype imputation, 6,539,982 SNPs remained for the analysis, which was performed by logistic regression models. The datasets went through a meta-analysis by the inverse variance weighted method under fixed effects. Main results and the role of chance Genetic associations with SCO at the genome-wide-level of significance were identified in the major histocompatibility (MHC) class II region (rs1136759, OR = 1.80, P = 1.32E-08) and in a regulatory region of chromosome 14 nearby the vaccinia-related kinase 1 (VRK1) gene (rs115054029, OR = 3.14, P = 4.37-08). VRK1 is a relevant proliferative factor for spermatogenesis that causes progressive loss of spermatogonia when disrupted in mouse models. The role of the MHC system in SCO susceptibility was comprehensively evaluated through a validated imputation method that infers classical MHC alleles and polymorphic amino acid positions. A serine at position 13 of the HLA-DRβ1 protein (defined by the risk allele of the lead variant rs1136759) explained most of the SCO association signals within the MHC class II region. This residue is located in the binding pocket of the HLA-DR molecule and interacts directly with the presented antigen. Interestingly, position 13 of HLA-DRβ1 is the most relevant risk amino acid position for a wide spectrum of immune-mediated disorders. The HLA-DRB1*13 haplotype (which includes the serine at position 13 and represents the strongest NOA-associated marker in Asians to date) was the strongest signal amongst the classical MHC alleles in our study cohort (OR = 1.93, P = 9.90E-07). Limitations, reasons for caution Although the statistical power for the overall analysis was appropriate, the subphenotype analyses performed had considerably lower counts, which may influence the identification of genetic variants conferring low to moderate risk effects. Independent studies in larger SCO study cohorts should be performed to confirm our findings. Wider implications of the findings The molecular mechanisms underlying unexplained SPGF are largely unknown. Our data suggest a relevant role of common genetic variation in the development of SCO, the most extreme histological phenotype of NOA. SCO is characterized by the loss of germ cells and, therefore, implies a considerably higher probability of unsuccessful TESE. Trial registration number N/A

Evaluation of differential serum expression of three factors and pulmonary function in patients with silicosis.

Silicosis is a chronic occupational lung disease. As was previously found by the authors, some proteins increased in the lung tissue of activated rats, and protein tyrosine phosphatase non-receptor type 2 (PTPN2), factor B, and vaccinia-related kinase 1 (VRK1) showed highly differential expressions. In this study, serum and bronchoalveolar lavage fluid samples were collected from patients with silicosis and healthy people to verify the expression of PTPN2, factor B, and VRK1. The diagnostic value of differentially expressed proteins for silicosis was judged. The expression levels of serum PTPN2, VRK1, and factor B in patients with silicosis were significantly higher than those in the control group (p < 0.01). Higher serum PTPN2 and factor B concentrations significantly and negatively correlated with the ratio of forced expiratory volume in 1 s to forced vital capacity (FEV1/FVC), maximum vital capacity (VCmax), FEV1, and FVC, suggesting that the high expression of PTPN2 and factor B is associated with decreased pulmonary ventilation function and restrictive ventilatory impairment in patients with silicosis. All area under curve (AUC) measurements generated from single detection events were >0.744, with PTPN2 reaching the highest value (0.858). The AUC, sensitivity, and specificity for the combined diagnosis using factor B and PTPN2 were 0.907, 86.91% and 85.07%, respectively, for factor B and PTPN2. The 3 differentially expressed proteins are potential classes of predictive biomarkers for silicosis. Regarding the economy and test practicality, the best diagnostic combination is factor B and PTPN2 for the analysis of AUC, sensitivity and specificity. Int J Occup Med Environ Health. 2021;34(4):527-40.

Adult-Onset Spinal Muscular Atrophy due to Mutations in the VRK1 Gene.

ObjectiveTo expand our knowledge of the range of clinical phenotypes associated with vaccinia-related kinase 1 (VRK1) gene mutations.MethodsWe present clinical and molecular data of 2 individuals with slowly progressive weakness and a clinical syndrome consistent with adult-onset spinal muscular atrophy without pontocerebellar atrophy.ResultsGenetic testing revealed likely pathogenic variants in the VRK1 gene in both subjects. One individual carried homozygous p.R321C (c.961 C>T), likely pathogenic variants. The other carried compound heterozygous p.V236M (c.706 G>A) and p.R321C (c.961 C>T), likely pathogenic variants. Notably, both patients were of Hispanic descent.ConclusionsWe report 2 cases with VRK1 mutations presenting as adult-onset spinal muscular atrophy without pontocerebellar hypoplasia and review the current literature of similar cases. Our report expands the clinical spectrum of neurologic disorders associated with VRK1 mutations.

Vaccinia-related kinase 2 drives pancreatic cancer progression by protecting Plk1 from Chfr-mediated degradation.

As a key cell cycle regulator, polo-like kinase 1 (Plk1) has been recognized as a crucial factor involved in the progression of pancreatic cancer (PC). However, its regulatory mechanism is poorly understood. Here, we present evidence that Plk1 is a novel substrate of vaccinia-related kinase 2 (VRK2), a serine-threonine kinase that is highly expressed and predicts poor prognosis in PC. VRK2 phosphorylates Plk1 at threonine 210 and protects it from ubiquitin-dependent proteasomal degradation. We showed that mechanistically complement factor H-related protein (CFHR), as a major E3 ligase, promotes Plk1 degradation by ubiquitinating it at lysine 209. Phosphorylation of Plk1 at threonine 210 by VRK2 interferes with the interaction of Chfr with Plk1 and antagonizes Plk1 ubiquitination, thereby stabilizing the Plk1 protein. Taken together, our data reveal a mechanism of Plk1 overexpression in PC and provide evidence for targeting VRK2 as a potential therapeutic strategy.

HNRNP A1 Promotes Lung Cancer Cell Proliferation by Modulating VRK1 Translation.

THeterogeneous nuclear ribonucleoprotein (HNRNP) A1 is the most abundant and ubiquitously expressed member of the HNRNP protein family. In recent years, it has become more evident that HNRNP A1 contributes to the development of neurodegenerative diseases. However, little is known about the underlying role of HNRNP A1 in cancer development. Here, we report that HNRNP A1 expression is significantly increased in lung cancer tissues and is negatively correlated with the overall survival of patients with lung cancer. Additionally, HNRNP A1 positively regulates vaccinia-related kinase 1 (VRK1) translation via binding directly to the 3′ untranslated region (UTR) of VRK1 mRNA, thus increasing cyclin D1 (CCND1) expression by VRK1-mediated phosphorylation of the cAMP response element–binding protein (CREB). Furthermore, HNRNP A1 binding to the cis-acting region of the 3′UTR of VRK1 mRNA contributes to increased lung cancer cell proliferation. Thus, our study unveils a novel role of HNRNP A1 in lung carcinogenesis via post-transcriptional regulation of VRK1 expression and suggests its potential as a therapeutic target for patients with lung cancer.

DNA Aptamers against Vaccinia-Related Kinase (VRK) 1 Block Proliferation in MCF7 Breast Cancer Cells.

Vaccinia-related kinase (VRK) 1 is a serin/threonine kinase that plays an important role in DNA damage response (DDR), phosphorylating some proteins involved in this process such as 53BP1, NBS1 or H2AX, and in the cell cycle progression. In addition, VRK1 is overexpressed in many cancer types and its correlation with poor prognosis has been determined, showing VRK1 as a new therapeutic target in oncology. Using in vitro selection, high-affinity DNA aptamers to VRK1 were selected from a library of ssDNA. Selection was monitored using the enzyme-linked oligonucleotide assay (ELONA), and the selected aptamer population was cloned and sequenced. Three aptamers were selected and characterized. These aptamers recognized the protein kinase VRK1 with an affinity in the nanomolar range and showed a high sensibility. Moreover, the treatment of the MCF7 breast cell line with these aptamers resulted in a decrease in cyclin D1 levels, and an inhibition of cell cycle progression by G1 phase arrest, which induced apoptosis in cells. These results suggest that these aptamers are specific inhibitors of VRK1 that might be developed as potential drugs for the treatment of cancer.

The C‐terminus of VRK1 Governs Interactions with Nucleosomes to Influence Histone H3 Threonine 3 (H3T3) Phosphorylation

Vaccinia-related kinase 1 (VRK1) is a highly abundant nuclear kinase that has numerous functions during mitosis, including the phosphorylation of the histone H3 tail at threonine 3 (H3T3ph). This post-translational modification, attributed to both VRK1 and the haspin kinase, is crucial for ensuring appropriate chromosome segregation. Despite the importance of VRK1 to cell cycle regulation, little is known about the biochemical basis of its activity on nucleosomes. Because placement of histone phosphorylation during mitosis is tightly spatiotemporally regulated, we sought to determine the chromatin template itself serves as a regulatory mechanism for VRK1 activity. In this work, we characterize the interaction between VRK1 and nucleosomes and determine how nucleosome binding affects nucleosome-directed kinase activity at H3T3. We find that VRK1 directly and stably interacts with nucleosomes with an apparent Kd of ~200 nM. VRK1 binds nucleosomes via a C-terminal alternating arginine motif, mutations or truncations of which are found in rare cases of genetic spinal muscular atrophy. Mutation or truncation of this motif leads to a 4-50 fold reduction in nucleosome affinity that is correlated with mutation position and severity. C-terminal arginine mutants of VRK1 show reduced capacity for histone H3T3 phosphorylation, suggesting that stable interaction with nucleosomes is required for nucleosome-directed kinase activity at H3T3. The nucleosome acidic patch, a negatively charged region at the dimer interface of H2A/H2B, is a hot spot for arginine-mediated nucleosome interactions. Neutralization of the acidic patch impairs VRK1 binding but surprisingly increases the activity of VRK1 at H3T3. In contrast, the presence of extended lengths of linker DNA promotes both nucleosome binding and phosphorylation of H3T3. Cryo-EM single particle analysis of VRK1-bound nucleosome particles demonstrates both acidic patch binding and heterogenous DNA binding that concentrates around the nucleosome linker DNA. This evidence suggests that VRK1 adopts at least two alternative binding modes on the nucleosome (acidic patch-governed vs. linker DNA-governed) that either promote or inhibit nucleosome-directed kinase activity based on the position and distance of VRK1 from the H3 tail. We suggest a model wherein dynamic access to the nucleosome acidic patch regulates the activity of VRK1 towards H3T3 during mitosis.

Vaccinia-related kinase 2 blunts sorafenib's efficacy against hepatocellular carcinoma by disturbing the apoptosis-autophagy balance.

Hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. Sorafenib is the only Food and Drug Administration (FDA)-approved first-line targeted drug for the treatment of advanced HCC. However, its effect on patient survival is limited. Recently, studies have demonstrated that the imbalance between apoptosis and autophagy plays a critical role in chemoresistance, and it is hypothesised that restoring the balance between these processes is a potential treatment strategy for improving chemoresistance in cancer. However, there is currently no evidence supporting this hypothesis. We aimed to investigate if vaccinia-related kinase 2 (VRK2), a serine/threonine protein kinase, confers sorafenib resistance in HCC cells. Here, we found that VRK2 was enriched in sorafenib-resistant HCC cells and patient-derived xenografts. Both in vivo and in vitro evidences showed that VRK2 blunts the efficacy of sorafenib against hepatocellular carcinoma by disturbing the balance between apoptosis and autophagy. Mechanistically, VRK2 promotes the phosphorylation of Bcl-2 by activating JNK1/MAPK8, thereby enhancing the dissociation of Bcl-2 from Beclin-1 and promoting the formation of the Beclin-1-Atg14-Vps34 complex, which facilitates autophagy. Furthermore, VRK2-induced phosphorylation of Bcl-2 promotes the interaction of Bcl-2 with BAX, thereby inhibiting apoptosis. In conclusion, targeting VRK2 for modulation of the balance between autophagy and apoptosis may be a novel strategy for overcoming sorafenib resistance in HCC.

Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners.

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix α1 and loop α1α2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between the phosphorylated residues and the positively charged C-terminal helix α6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a 5000-fold loss of affinity for dsDNA. However, it does not impair binding to lamin A/C Igfold domain and emerin nucleoplasmic region, which leaves open the question of the regulation of these interactions.

VRK2 inhibition synergizes with PD-1 blockade to improve T cell responses

Therapeutic programmed cell death protein 1 (PD-1) blockade enhances T cell mediated anti-tumor immunity but many patients do not respond and a significant proportion develops inflammatory toxicities. To develop better therapeutics and to understand the signaling pathways downstream of PD-1 we performed phosphoproteomic analysis of PD-1 and identified vaccinia related kinase 2 (VRK2) as a key mediator of PD-1 signaling. Using genetic and pharmacological approaches, we discovered that VRK2 is required for PD-1-induced phosphorylation of the protein p21 activated kinase 2 (PAK2), and for the inhibition of IL-2, IL-8, and IFN-γ secretion. Moving into in vivo syngeneic tumor models, pharmacologic inhibition of VRK2 in combination with PD-1 blockade enhanced tumor clearance through T cell activation. This study suggests that VRK2 is a unique therapeutic target and that combination of VRK2 inhibitors with PD-1 blockade may improve cancer immunotherapy.

VRK1 promotes proliferation, migration, and invasion of gastric carcinoma cells by activating β-catenin.

Vaccinia-related kinase 1 (VRK1) is a member of the VRK subfamily belonging to the casein kinase superfamily, and it regulates the proliferation and survival of cells both in normal and malignant tissues. A variety of transcription factors including c-Jun can also be specifically phosphorylated and stimulated by VRK1. However, the regulatory mechanism of VRK1 in gastric carcinoma (GC) remains unclear. This research aimed to determine the function of VRK1 during tumor progression in GC. The mRNA and protein expression of the VRK1 and other genes were evaluated in GC cell lines using real-time RT-PCR and western blotting. Cell proliferation was analyzed using the cell count kit-8 (CCK-8) assay, and cell migration and invasion were monitored by the Transwell assay. The downregulated genes in shVRK1 cells compared with shCtrl were assessed using RNA-seq. The interactions of VRK1 with β-catenin or c-Jun were detected by co-IP. We found that VRK1 was overexpressed in gastric cancer cells, conversely, knockdown of VRK1 inhibits GC cells' proliferation, migration, and invasion. Moreover, VRK1 might regulate the expression of β-catenin (CTNNB1) at the transcriptional level by phosphorylating c-Jun, the transcriptional factor of β-catenin. VRK1 changes the subcellular location and decreases the nuclear aggregation of c-Jun by phosphorylating the Ser243 site. To conclude, VRK1 can affect migration and invasion by regulating the expression of β-catenin at the transcriptional level in GC cells.

Genome-wide association study of vegetarianism in UK Biobank identifies association with VRK2

Prospective studies have observed differences in risks for several health outcomes when comparing meat-eaters and vegetarians, but the mechanisms underlying these differences remain uncertain. Identifying genetic factors related to vegetarianism may be valuable for assessing causality. We report a genome-wide association study (GWAS) of vegetarianism in 367,198 participants from UK Biobank. We identified one locus, rs10189138, near the vaccinia related kinase 2 (VRK2) gene, significantly associated with vegetarianism (β=0.153, p=3x10-8). The associations between rs10189138 and 40 traits were calculated, and the rs10189138 T allele (MAF=0.12) was found to be significantly associated with greater height, after controlling the false discovery rate (FDR). Correlations between genetically predicted vegetarianism and 855 other genetically predicted traits were also calculated, and vegetarianism had significant positive genetic correlations with fluid intelligence and age at menarche, after controlling the FDR. Future research on an independent sample is needed to see if this GWAS result can be replicated.

Downregulation of VRK1 reduces the expression of BANF1 and suppresses the proliferative and migratory activity of esophageal cancer cells.

Esophageal squamous cell carcinoma (ESCC) is a common malignancy worldwide. The disease has a poor prognosis and a low 5-year survival rate. Therefore, it is necessary to identify new strategies to optimize the treatment of ESCC. Vaccinia-related kinase (VRK1) and barrier-to-autointegration factor 1 (BANF1) are overexpressed in ESCC. In the present study, the roles of VRK1 and BANF1 were explored in the development of ESCC. In the present study, the effects of small interfering (si)RNA-induced downregulation of VRK1 on BANF1 expression were investigated as well as the effects on proliferative and migratory activity of ESCC cells. Western blot analysis indicated that the protein expression levels of BANF1 were decreased following siRNA depletion of VRK1. Furthermore, the depletion of VRK1 expression inhibited the proliferation and migration of ESCC cell lines, and flow cytometry analysis indicated that the depletion of VRK1 triggered cell cycle arrest mainly in the S phase. These results suggested that VRK1 and BANF1 may have pivotal roles in the progression of ESCC.

Phosphorylation of vaccinia-related kinase 1 at threonine 386 transduces glucose stress signal in human liver cells.

Vaccinia-related kinase 1 (VRK1) is a chromatin-associated Ser-Thr kinase that regulates numerous downstream factors including DNA repair as well as stress factors c-Jun and p53. Both c-Jun and p53 are phosphorylated at Ser63 and Thr18, respectively, in response to low glucose (40 mg/dl of medium) but not high glucose (140 mg/dl of medium) in human hepatoma-derived Huh-7 cells. Here, we have determined the molecular mechanism by which VRK1 phosphorylates these residues in response to glucose in Huh-7 cells. Human VRK1 auto-phosphorylates Ser376 and Thr386 in in vitro kinase assays. In Huh-7 cells, this auto-phosphorylation activity is regulated by glucose signaling; Thr386 is auto-phosphorylated only in low glucose medium, while Ser376 is not phosphorylated in either medium. A correlation of this low glucose response phosphorylation of Thr386 with the phosphorylation of c-Jun and p53 suggests that VRK1 phosphorylated at Thr386 catalyzes this phosphorylation. In fact, VRK1 knockdown by siRNA decreases and over-expression of VRK1 T386D increases phosphorylated c-Jun and p53 in Huh-7 cells. Phosphorylation by VRK1 of c-Jun but not p53 is regulated by cadherin Plakophilin-2 (PKP2). The PKP2 is purified from whole extracts of Huh-7 cells cultured in low glucose medium and is characterized to bind a C-terminal peptide of the VRK1 molecules to regulate its substrate specificity toward c-Jun. siRNA knockdowns show that PKP2 transduces low glucose signaling to VRK1 only to phosphorylate c-Jun, establishing the low glucose-PKP2-VRK1-c-Jun pathway as a glucose stress signaling pathway.

Nuclear Pregnane X Receptor Transduces Glucose Signal to Suppress Estrogen Sulfotransferase Gene in Human Liver Cells

Estrogen sulfotransferase (SULT1E1) metabolically inactivates estrogen and SULT1E1 expression is tightly regulated by multiple nuclear receptors. Previously, it was found that nuclear receptors HNF4a and RORa form as complexes binding to the SULT1E1 enhancer (−943/−922 bp) to activate gene expression in high glucose (450 mg/dL) treated human liver cells. However, the mechanism by which low glucose signal (40 mg/dL) mediated SULT1E1 suppression in human liver cells remain mysterious. Here, we have demonstrated that low glucose signal stimulates PXR phosphorylation at Ser350 on its ligand binding domain, preventing its heterodimerization with RXR and enabling it to interact with the HNF4a/RORa binding complexes on the enhancer, thereby suppressing SULT1E1 gene transcription. SULT1E1 was significantly repressed at both mRNA and protein levels in low glucose cultured human liver cells and this suppression was found to be PXR dependent. Vaccinia‐related kinase 1 (VRK1) was found to phosphorylate PXR at Ser350 in low glucose treated cells. Transient transfection analysis of SULT1E1 promoter‐luciferase reporter genes revealed that phosphorylated PXR significantly suppressed SULT1E1 promoter activity via the enhancer. Chromatin immunoprecipitation and gel shift assays elucidated that phosphorylated PXR bound to the SULT1E1 enhancer and disrupted the formation of the HNF4a/RORa binding complexes on it at low glucose condition. The physical interactions among these nuclear receptors were subsequently demonstrated by a series of co‐immunoprecipitation assays. Our study has shown that xenobiotic sensing nuclear receptor PXR transduces low glucose signal by its conserved phosphorylation to suppress SULT1E1 expression in human liver cells.Support or Funding InformationThis work was supported by National Institutes of Health intramural research program Z01ES1005‐01.